RESUMO
Many neurotransmitters and hormones signal by stimulating G protein-coupled neurotransmitter receptors (GPCRs), which activate G proteins and their downstream effectors. Whether these signalling proteins diffuse freely within the plasma membrane is not well understood. Recent studies have suggested that direct protein-protein interactions exist between GPCRs, G proteins and G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels. Here, we used fluorescence resonance energy transfer (FRET) combined with total internal reflection fluorescence microscopy to investigate whether proteins within this signalling pathway move within 100 A of each other in the plasma membrane of living cells. GABA(B) R1 and R2 receptors, Kir3 channels, Galphao subunits and regulators of G protein signalling (RGS4) proteins were each fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) and first assessed for functional expression in HEK293 cells. The presence of the fluorophore did not significantly alter the signalling properties of these proteins. Possible FRET was then investigated for different protein pair combinations. As a positive control, FRET was measured between tagged GABA(B) R1 and R2 subunits ( approximately 12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABA(B) R1 or R2 subunits ( approximately 13% FRET), and between Galphao and GABA(B) R1 or R2 subunits ( approximately 10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABA(B) R1 or R2 subunits ( approximately 10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Galphao. These data are discussed in terms of a model in which GABA(B) receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas RGS/fisiologia , Receptores de GABA-B/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , AMP Cíclico/metabolismo , Eletrofisiologia , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Interpretação de Imagem Assistida por Computador , Modelos Moleculares , Técnicas de Patch-Clamp , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Técnicas de Cultura de Tecidos , TransfecçãoRESUMO
Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (Galphai/o) G proteins. Although the Gbetagamma dimer is known to activate GIRK channels, the role of the Galphai/o subunit remains unclear. Here, we established that Galphao subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for Galphao binding. Further studies suggested that the Galphai/obetagamma heterotrimer binds to the GIRK2 C-terminal domain via Galpha and not Gbetagamma. Galphai/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and Gbetagamma-activated currents. Finally, PTX-insensitive Galphaq or Galphas subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive Galphai/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific Galphai/o signaling pathways.
